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Introduction

Researchers performing gene expression studies are often faced with the fact that samples are only available in limited quantity and quality. In this study, using M2 pyruvate kinase (M2PK) and beta-actin as targets, we investigated whether the LightCycler® RNA Pre-Amplification Kit can increase the sensitivity of qPCR assays for fragmented mRNA in research samples derived from individuals with pancreatic and colorectal cancer.

The LightCycler® RNA Pre-Amplification Kit provides an efficient method for isothermal amplification of total RNA. Only the original RNA transcripts are amplified (about 104 times, linearly), maintaining the relative representation of each species. The resulting cDNA pool can be used directly for subsequent applications.

Materials and Methods

cDNA conversion was carried out either by using a standard method with anchored oligo(dT)18 primers, or using the LightCycler® RNA Pre-Amplification Kit with both oligo(d)T and random primers. Primers and probes were designed by the ProbeFinder qPCR assay design software (available free for Roche´s Universal ProbeLibrary System). qPCR reactions were performed in triplicate on a Roche LightCycler® 480 Instrument using the provided UPL dual probe assay settings. The crossing-point (Cp) values for each amplification curve were calculated using the LightCycler® 480 Relative Quantification software.

Results and Discussion

In our experiments, the concentration of RNA extracted from pancreatic tissue samples with high RNAse activity was about 25.3 ng/µl. Using the LightCycler® RNA Pre-Amplification Kit, we produced a pool of 42 µl cDNA. Subsequent qPCR assays were performed using 0.5 µl of this pool in 20 µl total reaction volume. Obtained Cp values for ACTB and M2PK were 21.7 and 26.2, respectively. Further 10x dilution of the cDNA did not alter the difference of Cp values between the two genes (not shown). Thus, in our case, cDNA synthesized from 25 ng RNA with pre-amplification was sufficient for at least 84 PCR reactions, and, as shown by the dilutions done, potentially even up to 840 qPCR reactions. qPCR assays using the same volumes but a standard cDNA conversion method without amplification detected significantly higher Cp values for both genes from 300 ng total RNA (see Table 1).

Standard Method

RNA pre-Amplification method

Target

ACTB

M2PK

ACTB

M2PK

Sample

Mean Cp of Triplicates

STD Cp

Mean Cp of Triplicates

STD Cp

Mean Cp of Triplicates

STD Cp

Mean Cp of Triplicates

STD Cp

29.44

0.15

34.58

0.09

21.71

0.11

26.17

0.08

Table 1: Cp values of amplification curves of ACTB and M2PK from cDNA synthesized either by a standard method from 300 ng RNA, or the LightCycler® RNA Pre-Amplification Kit from 25 ng RNA extracted from pancreatic cancer tissue samples.

This research showed that highly significant Cp differences between both protocols were also obtained with cDNA from free-circulating plasma RNA obtained from individuals with colon cancer (see Figure 1).

Conclusions

The novel LightCycler® RNA Pre-Amplification Kit saves valuable RNA samples for multiple gene expression studies, and is also suitable for the pre-amplification of ultra-small and fragmented RNA samples extracted from research samples. Starting from, on average, 40 times less RNA than standard cDNA conversion assays, the produced cDNA is sufficient for up to 20 times more qPCR reactions at up to 1000 times higher sensitivity. This makes the kit extremely useful for gene expression analysis of very small sample amounts available in tissue banks as well as for amplification of very low-quantity and heavily fragmented RNA samples, such as free-circulating RNA isolated from peripheral blood.

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